This invention relates to flow cytometry and, more particularly, to applications of flow cytometry using single molecule identification. This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the U.S. Department of Energy. The government has certain rights in the invention.
Light-induced fluorescence detection of single molecules in liquid solution was first accomplished several years ago, and applications for single molecule detection (SMD) in the analytical, environmental, and biomedical sciences are beginning to emerge. Overviews of single molecule detection in solution are presented in, e.g., R. A. Keller et al., 50 Appl. Spectrosc., pp. A12-A32 (1996), and P. M. Goodwin et al., 29 Accounts Chem. Res., pp. 607-613 (1996). In general, an operable system will include a dilute stream of separated individual molecules with known fluorescent characteristics that are excited one at a time by a light source, where the resulting emitted photons are detected. One approach to single molecule detection used herein is based on flow cytometry, where the analyte solution is delivered into a rapidly flowing sheath fluid and hydrodynamically focused into a narrow sample stream. Yet another approach is based on analyte movement through a capillary, such as described in U.S. Pat. No. 5,209,834, issued May 11, 1993, to Shera, and incorporated herein by reference.
In flow cytometry, a sample stream passes through the center of a probe volume defined by the diameter of the focused excitation laser beam and a spatial filter placed in the image plane of a light collecting objective. Single fluorescent molecules are detected by the bursts of photons emitted as they flow through the detection volume one-at-a-time. Hydrodynamic focusing of the sample stream by the sheath fluid ensures that the entire sample stream passes through the center of the excitation laser so that single molecules delivered into the flow cell are detected with an efficiency exceeding 90%. See, e.g., P. M. Goodwin et al., "Progress toward DNA sequencing at the single molecule level," 41 Exp. Tech. Phys., pp. 279-294 (1995), incorporated herein by reference.
Some of the applications under development for efficient single molecule detection in flow include DNA fragment sizing, DNA sequencing, counting and sorting of single molecules, and detection of probe-target binding. A number of applications for SMD in solution require one to distinguish between different fluorophores present in a mixture. For example, in one approach to DNA sequencing (U.S. Pat. No. 4,962,037, issued Oct. 9, 1990, and incorporated herein by reference), each base is labeled with a different fluorescent probe, and a rapid, efficient method is needed to identify these fluorophores. In yet another approach, only two fluorescent probes are required (U.S. Pat. No. 5,405,747, issued Apr. 11, 1995, and incorporated herein by reference) to reduce the number of distinguishing characteristics that are required to be identified.
Several techniques have been developed to distinguish between different single molecules in solution. One technique employs two or more detection channels to identify single molecules in a multicomponent mixture based upon differences in excitation and emission wavelengths of the fluorophore labels. See, e.g., Soper et al., "Detection and identification of single molecules in solution," 9 J. Opt. Soc. Am. B, pp.1761-1769 (October 1992), incorporated herein by reference. Typically, the detection volume is probed by collinear laser beams at different wavelengths corresponding to the excitation maxima of each of the fluorophores. A beam splitter directs the fluorescence from each fluorophore to a separate filter/detector channel. This method requires that the emission bands of the fluorophores be sufficiently separated to minimize crosstalk between the respective channels. Furthermore, separating the fluorescence signal into distinct detection channels increases the complexity of the instrumentation and can reduce the overall detection efficiency.
An alternative approach that requires only a single detection channel and is applicable to molecules with similar spectroscopic properties exploits the difference in fluorescence lifetimes of the fluorophores. Single Rhodamine 6G (R6G) molecules in flow have been distinguished from Rhodamine B (RB) molecules and from tetramethyl rhodamine isothiocyanate (TRITC) molecules by applying a maximum likelihood function to time-correlated single-photon counting (TCSPC) fluorescence decay measurements of individual bursts in a mixed sample (Zander et al.,63 Appl. Phys. B, pp. 517-523 (1996); J. Enderlein et al., 270 Chem. Phys. Lett., pp. 464-470 (1997)). Also, a rhodamine derivative JA169 has been distinguished from a carbocyanine dye Cy5 using this technique (M. Sauer et al., 65 Appl. Phys. B. No. 3, pp. 427-431 (August 1997).
Objects, advantages and novel features of the invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon examination of the following or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.